Organic Cation Transporters (OCTs) influence the plasma concentration of many endogenous molecules, and of an even wider array of xenobiotic compounds (incl. pharmacologically and toxicologically active agents). Although expressed in many tissues, the highest levels of expression are typically found in barrier epithelia, including the kidney, liver and intestine where they influence drug bioavailability; drug resistance; excretion of drugs and their metabolites; drug toxicity; and drug pharmacokinetics and pharmacodynamics. Thus, OCT transporters play a critical role in fundamental cellular processes in health and disease, and function as important mediators governing all aspects of drug therapy. Despite the apparent physiological and clinical importance of OCT proteins, knowledge of their structure and mechanism of action has lagged far behind the knowledge of these properties of proteins in general. In this proposal, we outline four Specific Aims that will apply a series of experimental and computational approaches to address the structure of a prototypical organic cation transporter, OCT2: (1) Define the membrane topology of OCT transporters using a series of topology scanning approaches; (2) Define the functional regions of OCTs by site-directed mutagenesis; (3) Construct a comprehensive structural and predictive model of OCT2 that can correlate structural point mutations to changes in substrate affinity and transport," and (4) Determine the substrate binding domains of OCT transporters by mass spectrometry employing selective photoaffinity labels to determine peptide sequences structurally associated with the binding site. The results will be used to develop a model of the structural factors that influence binding of substrates to human OCT2. Application of the information gained through these studies holds the promise of predicting potential drug-interactions and the basis of genetic differences in renal secretion of cationic xenobiotics.